An Integrated Imaging System for High-throughput Nanoscopy of the 4D Nucleome

 描述(申请人提供)：DNA、蛋白质和RNA的复合体称为染色质，是基本细胞过程的底物，如基因转录、调节、复制和修复。因此，如果我们要了解这些过程的机制及其在发育和疾病中的影响，就必须解开它的结构和动力学。然而，染色质是一个很难研究的目标：它存在于核内拥挤的环境中，在结构上有多个长度尺度，在核内和核之间可变，并且具有高度的动态性。捕捉这些信息需要仪器可以(I)测量多个长度尺度，从整个细胞到几十纳米，(Ii)跟踪活细胞中的染色质动态，以及(Iii)在可管理的时间框架内获取并量化数千幅图像，以克服内在的变异性，并提供染色质行为的统计描述。这样的仪器还不存在，现有的仪器在分辨率、动态速度和吞吐量方面受到限制。我们提出了一种创新的多学科方法，结合光学、数据处理和建模的发展，实现了一个自动化的高通量超分辨率成像和细胞核内致密单分子跟踪的集成系统。我们的具体目标是：1)开发一种用于核内动态成像和粒子跟踪的自动化多色3D单分子开关(SMS)纳米显微镜，2)开发用于100-1,000个细胞/小时的高通量3D-SMS纳米检查的数据处理工具，3)开发染色质建模工具，该工具利用高通量粒子跟踪和SMS纳米检查提供的4D数据的前所未有的详细程度和统计深度，以及4)通过将它们应用于染色质体系结构领域的各种具有代表性的重要问题，包括转录因子和核小体的流动性和动力学，来测试其性能并改进我们的技术开发，以及联会组装和端粒重组的过程。这项提议从根本上背离了纳米图像生成过程的传统观点，即将专家用户大量参与的动手过程转变为注重量化和效率的自动化、高通量方法。通过使数万个细胞的纳米研究成为可能，我们预计我们的仪器将首次使核小体的时空动力学能够被定量地研究到单个核小体的水平。

 DESCRIPTION (provided by applicant): The complex of DNA, protein, and RNA known as chromatin is the substrate for essential cellular processes such as gene transcription, regulation, replication, and repair. Unravelling its structure and dynamics is therefore essential f we are to understand the mechanics of these processes and their effects in development and disease. Chromatin is, however, a difficult target to study: it is found in a crowded environment within the nucleus, is structurally organized on multiple length scales, is variable within and between nuclei, and is highly dynamic. Capturing this information requires instrumentation which can (i) measure on multiple length scales, from the whole cell down to tens of nm, (ii) follow chromatin dynamics in living cells, and (iii) acquire and quantify thousands of images in a manageable time frame to overcome the intrinsic variability and provide a statistical description of chromatin behavior. Such an instrument does not yet exist, with existing instrumentation being limited in resolution, dynamic speed, and throughput. We propose an innovative multi-disciplinary approach that combines developments in optics, data processing and modeling to realize an integrated system for automated high-throughput super- resolution imaging and dense single-molecule tracking in the cell nucleus. Our Specific Aims are: 1) Develop an automated multicolor 3D single-molecule switching (SMS) nanoscope for dynamic imaging and particle-tracking in the nucleus, 2) Develop data processing tools for high-throughput 3D- SMS nanoscopy of 100-1,000 cells/h, 3) Develop chromatin modeling tools that take advantage of the unprecedented level of detail and statistical depth of the 4D data provided by high-throughput particle- tracking and SMS nanoscopy, and 4) test the performance and refine our technical developments by applying them to a diverse set of representative and important questions in the field of chromatin architecture, including the mobility and dynamics of transcription factors and nucleosomes, and the processes of synaptonemal assembly and telomere recombination. The proposal represents a fundamental departure from the traditional view of the nanoscopy image generation procedure as a hands-on process heavily involving an expert user to an automated, high- throughput method with focus on quantification and efficiency. By making nanoscopy studies of tens of thousands of cells feasible, we anticipate that our instrument will enable, for the first time, the spatiotemporal dynamics of the nucleome to be quantitatively investigated down to the single nucleosome level.